RESUMEN
Accumulating evidence implicates that herpes simplex virus type 1 (HSV-1) has been linked to the development and progression of Alzheimer's disease (AD). HSV-1 infection induces ß-amyloid (Aß) deposition in vitro and in vivo, but the effect and precise mechanism remain elusive. Here, we show that HSV-1 infection of the brains of transgenic 5xFAD mice resulted in accelerated Aß deposition, gliosis, and cognitive dysfunction. We demonstrate that HSV-1 infection induced the recruitment of microglia to the viral core to trigger microglial phagocytosis of HSV-GFP-positive neuronal cells. In addition, we reveal that the NLRP3 inflammasome pathway induced by HSV-1 infection played a crucial role in Aß deposition and the progression of AD caused by HSV-1 infection. Blockade of the NLRP3 inflammasome signaling reduces Aß deposition and alleviates cognitive decline in 5xFAD mice after HSV-1 infection. Our findings support the notion that HSV-1 infection is a key factor in the etiology of AD, demonstrating that NLRP3 inflammasome activation functions in the interface of HSV-1 infection and Aß deposition in AD.
Asunto(s)
Enfermedad de Alzheimer , Progresión de la Enfermedad , Herpesvirus Humano 1 , Ratones Transgénicos , Microglía , Proteína con Dominio Pirina 3 de la Familia NLR , Fagocitosis , Transducción de Señal , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/virología , Ratones , Microglía/metabolismo , Microglía/patología , Microglía/virología , Transducción de Señal/fisiología , Humanos , Herpes Simple/patología , Herpes Simple/inmunología , Herpes Simple/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animales de Enfermedad , Presenilina-1/genética , Presenilina-1/metabolismoRESUMEN
The pathogenesis of Parkinson's disease (PD) has been associated with mitochondrial dysfunction. Given that the PINK1/Parkin pathway governs mitochondrial quality control by inducing mitophagy to remove damaged mitochondria, therapeutic approaches to activate PINK1/Parkin-mediated mitophagy have the potential in the treatment of PD. Here, we have identified a new small molecule, BL-918, as an inducer of mitophagy via activating the PINK1/Parkin pathway. BL-918 triggers PINK1 accumulation and Parkin mitochondrial translocation to initiate PINK1/Parkin-mediated mitophagy. We found that mitochondrial membrane potential and mitochondrial permeability transition pore were involved in BL-918-induced PINK1/Parkin pathway activation. Moreover, we showed that BL-918 mitigated PD progression in MPTP-induced PD mice in a PINK1-dependent manner. Our results unravel a new activator of the PINK1/Parkin signaling pathway and provide a potential strategy for the treatment of PD and other diseases with dysfunctional mitochondria.
Asunto(s)
Mitocondrias , Mitofagia , Enfermedad de Parkinson , Proteínas Quinasas , Transducción de Señal , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Animales , Ratones , Mitofagia/efectos de los fármacos , Humanos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/genética , Mitocondrias/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Masculino , Progresión de la Enfermedad , FenilacetatosRESUMEN
The activation of stimulator of interferon genes (STING) signaling induces the production of type I interferons (IFNs), which play critical roles in protective innate immunity for the host to defend against viral infections. Therefore, achieving sustained or enhanced STING activation could become an antiviral immune strategy with potential broad-spectrum activities. Here, we discovered that various clinically used microtubule-destabilizing agents (MDAs) for the treatment of cancer showed a synergistic effect with the activation of STING signaling in innate immune response. The combination of a STING agonist cGAMP and a microtubule depolymerizer MMAE boosted the activation of STING innate immune response and showed broad-spectrum antiviral activity against multiple families of viruses. Mechanistically, MMAE not only disrupted the microtubule network, but also switched the cGAMP-mediated STING trafficking pattern and changed the distribution of Golgi apparatus and STING puncta. The combination of cGAMP and MMAE promoted the oligomerization of STING and downstream signaling cascades. Importantly, the cGAMP plus MMAE treatment increased STING-mediated production of IFNs and other antiviral cytokines to inhibit viral propagation in vitro and in vivo. This study revealed a novel role of the microtubule destabilizer in antiviral immune responses and provides a previously unexploited strategy based on STING-induced innate antiviral immunity.
Asunto(s)
Interferón Tipo I , Proteínas de la Membrana , Proteínas de la Membrana/genética , Inmunidad Innata , Transducción de Señal , Citocinas , Interferón Tipo I/farmacologíaRESUMEN
Lipid metabolism has been associated with the cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) stimulator of interferon genes (STING) DNA-sensing pathway, but our understanding of how these signals are integrated into a cohesive immunometabolic program is lacking. Here, we have identified liver X receptor (LXR) agonists as potent inhibitors of STING signaling. We show that stimulation of lipid metabolism by LXR agonists specifically suppressed cyclic GMP-AMP (cGAMP)-STING signaling. Moreover, we developed cyclic dinucleotide-conjugated beads to biochemically isolate host effectors for cGAMP inhibition, and we found that LXR ligands stimulated the expression of sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A), which is a 2'3'-cGAMP-degrading enzyme. Results of crystal structures suggest that cGAMP analog induces dimerization of SMPDL3A, and the dimerization is critical for cGAMP degradation. Additionally, we have provided evidence that SMPDL3A cleaves cGAMP to restrict STING signaling in cell culture and mouse models. Our results reveal SMPDL3A as a cGAMP-specific nuclease and demonstrate a mechanism for how LXR-associated lipid metabolism modulates STING-mediated innate immunity.
Asunto(s)
Metabolismo de los Lípidos , Nucleotidiltransferasas , Animales , Ratones , Receptores X del Hígado/metabolismo , Nucleotidiltransferasas/metabolismo , ADN , Nucleótidos Cíclicos/metabolismo , Inmunidad InnataRESUMEN
2'3'-cyclic GMP-AMP (2'3'-cGAMP) and 3'2'-cGAMP activate STING-dependent antiviral immunity in Drosophila melanogaster but fail to control infection by C virus in some fly species. In this issue of Immunity, Cai et al. reveal that Drosophila produces multiple cyclic di-nucleotides (CDNs) in response to viral infection. One of these CDNs-2'3'-c-di-GMP-is a very potent STING activator capable of promoting antiviral immunity in otherwise susceptible flies.
Asunto(s)
Drosophila melanogaster , Sistemas de Mensajero Secundario , Animales , Transducción de Señal , Antivirales , DrosophilaRESUMEN
DNA derived from chemotherapeutics-killed tumor cells is one of the most important damage-associated molecular patterns that can activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway in antigen-presenting cells (APCs) and promote antitumor immunity. However, conventional chemotherapy displays limited tumor cell killing and ineffective transfer of stable tumor DNA to APCs. Here we show that liposomes loaded with an optimized ratio of indocyanine green and doxorubicin, denoted as LID, efficiently generate reactive oxygen species upon exposure to ultrasound. LID plus ultrasound enhance the nuclear delivery of doxorubicin, induce tumor mitochondrial DNA oxidation, and promote oxidized tumor mitochondrial DNA transfer to APCs for effective activation of cGAS-STING signaling. Depleting tumor mitochondrial DNA or knocking out STING in APCs compromises the activation of APCs. Furthermore, systemic injection of LID plus ultrasound over the tumor lead to targeted cytotoxicity and STING activation, eliciting potent antitumor T cell immunity, which upon the combination with immune checkpoint blockade leads to regression of bilateral MC38, CT26, and orthotopic 4T1 tumors in female mice. Our study sheds light on the importance of oxidized tumor mitochondrial DNA in STING-mediated antitumor immunity and may inspire the development of more effective strategies for cancer immunotherapy.
Asunto(s)
ADN Mitocondrial , Liposomas , Femenino , Animales , Ratones , Mitocondrias , Inmunoterapia , ADN de Neoplasias , Cromogranina A , Doxorrubicina/farmacologíaRESUMEN
Cyclic GMP-AMP (cGAMP) is a second messenger that activates the stimulator of interferon genes (STING) innate immune pathway to induce the expression of type I IFNs and other cytokines. Pharmacologic activation of STING is considered a potent therapeutic strategy in cancer. In this study, we used a cell-based phenotypic screen and identified podophyllotoxin (podofilox), a microtubule destabilizer, as a robust enhancer of the cGAMP-STING signaling pathway. We found that podofilox enhanced the cGAMP-mediated immune response by increasing STING-containing membrane puncta and the extent of STING oligomerization. Furthermore, podofilox changed the trafficking pattern of STING and delayed trafficking-mediated STING degradation. Importantly, the combination of cGAMP and podofilox had profound antitumor effects on mice by activating the immune response through host STING signaling. Together, these data provide insights into the regulation of cGAMP-STING pathway activation and demonstrate what we believe to be a novel approach for modulating this pathway and thereby promoting antitumor immunity.
Asunto(s)
Neoplasias , Podofilotoxina , Animales , Ratones , Podofilotoxina/farmacología , Proteínas de la Membrana/metabolismo , Transducción de Señal , Inmunidad InnataRESUMEN
It is widely known that stimulator of interferon genes (STING) can trigger nuclear factor κB (NF-κB) signaling. However, whether and how the NF-κB pathway affects STING signaling remains largely unclear. Here, we report that Toll-like receptor (TLR)-, interleukin-1 receptor (IL-1R)-, tumor necrosis factor receptor (TNFR)-, growth factor receptor (GF-R)-, and protein kinase C (PKC)-mediated NF-κB signaling activation dramatically enhances STING-mediated immune responses. Mechanistically, we find that STING interacts with microtubules, which plays a crucial role in STING intracellular trafficking. We further uncover that activation of the canonical NF-κB pathway induces microtubule depolymerization, which inhibits STING trafficking to lysosomes for degradation. This leads to increased levels of activated STING that persist for a longer period of time. The synergy between NF-κB and STING triggers a cascade-amplified interferon response and robust host antiviral defense. In addition, we observe that several gain-of-function mutations of STING abolish the microtubule-STING interaction and cause abnormal STING trafficking and ligand-independent STING autoactivation. Collectively, our data demonstrate that NF-κB activation enhances STING signaling by regulating microtubule-mediated STING trafficking.
Asunto(s)
FN-kappa B , Transducción de Señal , Interferones , FN-kappa B/metabolismo , Transducción de Señal/genética , Receptores Toll-Like , Proteínas de la MembranaRESUMEN
Oxysterol-binding protein (OSBP) and its related proteins (ORPs) are a family of lipid transfer proteins (LTPs) that mediate non-vesicular lipid transport. ORP9 and ORP10, members of the OSBP/ORPs family, are located at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCSs). It remained unclear how they mediate lipid transport. In this work, we discovered that ORP9 and ORP10 form a binary complex through intermolecular coiled-coil (CC) domain-CC domain interaction. The PH domains of ORP9 and ORP10 specially interact with phosphatidylinositol 4-phosphate (PI4P), mediating the TGN targeting. The ORP9-ORP10 complex plays a critical role in regulating PI4P levels at the TGN. Using in vitro reconstitution assays, we observed that while full-length ORP9 efficiently transferred PI4P between two apposed membranes, the lipid transfer kinetics was further accelerated by ORP10. Interestingly, our data showed that the PH domains of ORP9 and ORP10 participate in membrane tethering simultaneously, whereas ORDs of both ORP9 and ORP10 are required for lipid transport. Furthermore, our data showed that the depletion of ORP9 and ORP10 led to increased vesicle transport to the plasma membrane (PM). These findings demonstrate that ORP9 and ORP10 form a binary complex through the CC domains, maintaining PI4P homeostasis at ER-TGN MCSs and regulating vesicle trafficking.
Asunto(s)
Retículo Endoplásmico , Fosfatos de Fosfatidilinositol , Transporte Biológico , Membrana Celular , Red trans-Golgi/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Double-stranded DNA (dsDNA) is recognized as a danger signal by cyclic GMP-AMP synthase (cGAS), which triggers innate immune responses. cGAS activity must be properly regulated to maintain immune homeostasis. However, the mechanism by which cGAS activation is controlled remains to be better understood. In this study, we identified USP15 as a cGAS-interacting partner. USP15 promoted DNA-induced cGAS activation and downstream innate immune responses through a positive feedback mechanism. Specifically, USP15 deubiquitylated cGAS and promoted its activation. In the absence of DNA, USP15 drove cGAS dimerization and liquid condensation through the USP15 intrinsic disordered region (IDR), which prepared cGAS for a rapid response to DNA. Upon DNA stimulation, USP15 was induced to express and boost cGAS activation, functioning as an efficient amplifier in innate immune signal transduction. In summary, the positive role played by USP15-mediated cGAS activation may be a novel regulatory mechanism in the fine-tuning of innate immunity.
Asunto(s)
Inmunidad Innata , Nucleotidiltransferasas , Nucleotidiltransferasas/metabolismo , Inmunidad Innata/fisiología , ADN/genética , Transducción de Señal/genéticaRESUMEN
Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.
Asunto(s)
Factores de Restricción Antivirales , Catelicidinas , Inmunidad Innata , Interferones , Factores de Restricción Antivirales/inmunología , Catelicidinas/inmunología , Humanos , Interferones/inmunología , Proteínas de la Membrana/metabolismo , Nucleótidos CíclicosRESUMEN
For new principal investigators, the first years are key to getting a laboratory off the ground and running. COVID-19 has changed the world, bringing on unforeseen difficulties and challenges at every level. We asked these investigators to share their experiences in navigating the unique environment since the start of the pandemic-what has changed in their vision for their laboratory, how they have adapted, and what advice they can share with others in a similar situation.
Asunto(s)
COVID-19/epidemiología , Laboratorios , Adaptación Psicológica , Investigación Biomédica/tendencias , COVID-19/psicología , Comunicación , Humanos , Laboratorios/tendencias , Personal de Laboratorio/psicología , Personal de Laboratorio/tendencias , SARS-CoV-2RESUMEN
The DNA-sensing enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) regulates inflammation and immune defense against pathogens and malignant cells. Although cGAS has been shown to exert antitumor effects in several mouse models harboring transplanted tumor cell lines, its role in tumors arising from endogenous tissues remains unknown. Here, we show that deletion of cGAS in mice exacerbated chemical-induced colitis and colitis-associated colon cancer (CAC). Interestingly, mice lacking cGAS were more susceptible to CAC than those lacking stimulator of interferon genes (STING) or type I interferon receptor under the same conditions. cGAS but not STING is highly expressed in intestinal stem cells. cGAS deficiency led to intestinal stem cell loss and compromised intestinal barrier integrity upon dextran sodium sulfate-induced acute injury. Loss of cGAS exacerbated inflammation, led to activation of STAT3, and accelerated proliferation of intestinal epithelial cells during CAC development. Mice lacking cGAS also accumulated myeloid-derived suppressive cells within the tumor, displayed enhanced Th17 differentiation, but reduced interleukin (IL)-10 production. These results indicate that cGAS plays an important role in controlling CAC development by defending the integrity of the intestinal mucosa.
Asunto(s)
Neoplasias del Colon/enzimología , Mucosa Intestinal/enzimología , Proteínas de Neoplasias/metabolismo , Nucleotidiltransferasas/metabolismo , Animales , Neoplasias del Colon/genética , Ratones , Ratones Noqueados , Células Supresoras de Origen Mieloide/enzimología , Proteínas de Neoplasias/genética , Nucleotidiltransferasas/genética , Células Madre/enzimología , Células Th17/enzimologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
Asunto(s)
Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Animales , Retículo Endoplásmico/inmunología , Humanos , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares , Transporte de Proteínas/fisiologíaRESUMEN
The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system1. Cyclic GMP-AMP synthase (cGAS) is a sensor of cytoplasmic DNA that activates the type-I interferon pathway2. On binding to DNA, cGAS is activated to catalyse the synthesis of cyclic GMP-AMP (cGAMP) from GTP and ATP3. cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING)3-9. STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines10,11. However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a ß-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Pollos , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Nucleótidos Cíclicos/metabolismo , Fosforilación , Unión Proteica/genética , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS)1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses.
Asunto(s)
Pollos , Microscopía por Crioelectrón , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Nucleótidos Cíclicos/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/química , Modelos Moleculares , Nucleótidos Cíclicos/químicaRESUMEN
A critical function of the PTEN-induced kinase 1 (PINK1)-Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant Tripterygium wilfordii, blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90). Celastrol's effect on mitophagy was independent of its known role in microtubule disruption. Instead, we show that celastrol suppresses Parkin recruitment by inactivating PINK1 and preventing it from phosphorylating Parkin and also ubiquitin. We also observed that PINK1 directly and strongly associates with TOM20, a component of the translocase of outer mitochondrial membrane (TOM) machinery and relatively weak binding to another TOM subunit, TOM70. Moreover, celastrol disrupted binding between PINK1 and TOM20 both in vitro and in vivo but did not affect binding between TOM20 and TOM70. Using native gel analysis, we also show that celastrol disrupts PINK1 complex formation upon mitochondrial depolarization and sequesters PINK1 to high-molecular-weight protein aggregates. These results reveal that celastrol regulates the mitochondrial quality control pathway by interfering with PINK1-TOM20 binding.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mitofagia/efectos de los fármacos , Proteínas Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Tripterygium/química , Triterpenos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Células HeLa , Humanos , Hidrazonas/antagonistas & inhibidores , Hidrazonas/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Triterpenos Pentacíclicos , Polimerizacion , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Dibenzothiophene (DBT) and their derivatives, accounting for the major part of the sulfur components in crude oil, make one of the most significant pollution sources. The DBT sulfone monooxygenase BdsA, one of the key enzymes in the "4S" desulfurization pathway, catalyzes the oxidation of DBT sulfone to 2'-hydroxybiphenyl 2-sulfonic acid (HBPSi). Here, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. BdsA and the BdsA-FMN complex exist as tetramers. DBT sulfone was placed into the active site by molecular docking. Seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are found to be involved in the binding of DBT sulfone. The importance of these residues is supported by the study of the catalytic activity of the active site variants. Structural analysis and enzyme activity assay confirmed the importance of the right position and orientation of FMN and DBT sulfone, as well as the involvement of Ser139 as a nucleophile in catalysis. This work combined with our previous structure of DszC provides a systematic structural basis for the development of engineered desulfurization enzymes with higher efficiency and stability.
RESUMEN
Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m-chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib.